N acetyl d glucosamine

Medium molecular weight chitosan from the exoskeleton of Lipopenaeus vannamei shrimp (Mv = 270 kDa) determined by viscometry (PSL Rheotek, São Paulo, Brazil) [41 (link)], and the degree of deacetylation (DD = 88%) determined by the infrared spectroscopy (Perkin Elmer, Beaconsfield, U.K.) method [41 (link)] were produced in the Northeastern Biomaterials Evaluation and Development Laboratory—CERTBIO (Campina Grande, PB, Brazil). This chitosan (CS) is accredited by the National Institute of Metrology, Quality and Technollogy (INMETRO) at the International Organization of Standardization (ISO)/International Electrotechnical Commission (IEC) 17025:2005, and is used for medical applications. Lactic acid was obtained from Vetec^® (Duque de Caixas/Rio de Janeiro, Brazil). Sodium hydroxide (NaOH) and methanol (CH₃OH) were purchased from Neon^® (São Paulo, Brazil). N-acetyl-D-glucosamine (>99%), phosphate buffered saline (PBS), and lysozyme (hen egg-white—HEW) were purchased from Sigma-Aldrich^® (Darmstadt, Germany).

Myoglobin from equine skeletal muscle (95–100% purity, lyophilized powder), N-acetyl-D-glucosamine (≥99% purity), D-glucose (≥99.5% purity), D-glucosamine hydrochloride (≥99% purity), potassium phosphate monobasic and dibasic, sodium azide, HPLC-grade solvents (acetonitrile, methanol, formic acid, trifluoroacetic acid), glucosone (2-keto-D-glucose; ≥98.0% purity; M_W 178.14 Da), glyoxal (ethanedial; 40% in H₂O; M_W 58.04 Da), methylglyoxal (2-oxopropanal; 40% in H₂O; M_W 72.06 Da), diacetyl (butane-2,3-dione; ≥95.0% purity; M_W 86.09 Da), 1,2-diaminobenzene, DTPA and Thioflavin T were purchased from Sigma-Aldrich (St. Louis, MO). 3-deoxyglucosone (3-Deoxy-D-erythro-hexosulose; ≥95% purity; M_W 162.14 Da) was obtained from Cayman Chemical (Ann Arbor, MI). SPE tC-18 Sep-Pak Vac 6 cc columns were obtained from Waters (Milford, MA). Filtration membranes (0.22 μm) were from Millipore (Billerica, MA). 3,5 dimethoxy-4-hydroxycinnamic acid (sinapinic acid), ProMix3 (mass spectrometry protein standards) were obtained from LaserBiolabs. Standard ion calibration solution (Pierce LTQ) for electrospray ionization in positive mode was purchased from Thermo Scientific (Rockford, IL, USA). Milli-Q purified distilled water for buffers and reagents preparations were purchased from Waters Millipore (Milford, MA, USA). All the reagents and chemicals used in the study were of analytical grade.

Carbohydrate inhibition assays were followed as mentioned by Chen et al. (24 (link)). The sugar specificities of cockroach lectins were investigated by competitive binding using the following carbohydrates: D-(+)-glucose, D-(+)-galactose, D-(+)-mannose, L-(−)-fucose, lactose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, fructose and arabinose (all purchased from Sigma Co.). Stock solutions of sugars were prepared in PBS at 0.2M and stored in −4 °C until use. Two folded serial dilutions of haemolymph (each of 5μl) were prepared in PBS followed by addition of 5μl of appropriate carbohydrate at the above initial concentration. The plates were incubated at room temperature for 60min and then 5μl of the mouse RBCs added to the respective wells. The controls were consisted of carbohydrate plus PBS, and plus RBCs alone. Inhibitory effects were recorded as those reducing agglutination in the wells. The end points of agglutination were examined under a stereomicroscope and by the flow characteristics of the erythrocyte pellets when the plate was held at an angle. This experiment was replicated three times.

To induce hypha formation, C. albicans strains were grown for 12 h in YPD, diluted to an OD₆₀₀ of 0.3 to 0.4 in RPMI or yeast nitrogen base (YNB) containing 1.25% N-acetyl-d-glucosamine (GlcNAc; Sigma), and incubated for 2 to 4 h at 37°C. Both RPMI and YNB+GlcNAc were supplemented with 0.1% 2-deoxyglucose (2-DG), 0.2% sodium acetate, or 0.2% sodium acetate with 5 mM F1,6-BP. Images were acquired using bright-field microscopy with a Zeiss Axio Observer Z1 microscope at a magnification of ×10. Hyphal formation was determined in multiple fields from at least 100 cells, and percent germination was calculated as the number of germinated cells relative to total observed C. albicans cells.